Organic compounds

ABSTRACT

The present invention relates to isolated polypeptides which comprise an amino acid sequence consisting of a mutated functional Abl kinase domain, said mutated functional kinase domain being resistant to inhibition of its tyrosine kinase activity by N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-piperazin-1-ylmethyl)-benzamide or a salt thereof, to the use of such polypeptides to screen for compounds which inhibit the tyrosine kinase activity of such polypeptides, to nucleic acid molecules encoding such polypeptides, to recombinant vectors and host cells comprising such nucleic acid molecules and to the use of such nucleic acid molecules in the production of such polypeptides for use in screening for compounds which inhibit the tyrosine kinase activity of such polypeptides.

FIELD OF THE INVENTION

[0001] This invention relates to isolated polypeptides which comprise anamino acid sequence consisting of a mutated functional Abl kinasedomain, said mutated functional kinase domain being resistant toinhibition of its tyrosine kinase activity byN-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-piperazin-1-ylmethyl)-benzamideor a salt thereof, to the use of such polypeptides to screen forcompounds which inhibit the tyrosine kinase activity of suchpolypeptides, to nucleic acid molecules encoding such polypeptides, torecombinant vectors and host cells comprising such nucleic acidmolecules and to the use of such nucleic acid molecules in theproduction of such polypeptides for use in screening for compounds whichinhibit the tyrosine kinase activity of such polypeptides.

BACKGROUND OF THE INVENTION

[0002] Bcr-Abl, a constitutively activated tyrosine kinase resultingfrom the formation of the Philadelphia chromosome [Nowell P. C. andHungerford D. A., Science 132, 1497 (1960)] by reciprocal translocationbetween the long arms of chromosomes 9 and 22 [Rowley J. D., Nature 243,290-293 (1973)], has been established as the characteristic molecularabnormality present in virtually all cases of chronic myeloid leukemia(CML) and up to 20 percent of adult acute lymphoblastic leukemia (ALL)[Faderl S. et al., N Engl J Med 341, 164-172 (1999); Sawyers C. L., NEngl J Med 340, 1330-1340 (1999)]. Bcr-Abl is sufficient to cause CML inmice [Daley G. Q. et al., Science 247, 824-830 (1990)] and itstransforming capacity is absolutely dependent on tyrosine kinaseactivity [Lugo T. G. et al., Science 247, 1079 (1990)]. The compoundN-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-piperazin-1-ylmethyl)-benzamide(hereinafter also referred to as “STI571”; STI571 is described in EP 0564 409 and, in the form of the methane sulfonate salt, in WO 99/03854),a competitive inhibitor at the ATP-binding site of Bcr-Abl, as well asof the receptor for platelet-derived growth factor, and c-kit tyrosinekinase [Lugo T. G. et al., Science 247, 1079 (1990)], has been shown tobe capable of very rapidly reversing the clinical and hematologicalabnormalities of CML in chronic phase and in blast crisis as well as ofPh-chromosome-positive (Ph+) acute lymphoblastic leukemia (Ph+ALL)[Druker B. J. et al., N Engl J Med 344, 1031-1037 (2001); Druker B. J.et al., N Engl J Med 344, 1038-1042 (2001)]. Whereas almost all chronicphase CML patients durably respond, remissions in CML blast crisis andPh+ALL are transient, and most patients relapse after several months,despite continued therapy with STI571 [Druker B. J. et al., N Engl J Med344, 1038-1042 (2001)]. The mechanism of resistance to STI571 is subjectof intense research.

[0003] I was now surprisingly found that mutations present in the kinasedomain of the Bcr-Abl gene of patients suffering from CML or Ph+ALLaccount for the biological resistance of these patients towards STI571treatment in that said mutations lead to resistance of the Bcr-Abltyrosine kinase towards inhibition by STI571.

[0004] These findings are extremely valuable in e.g. finding newcompounds or combinations of compounds which are capable to overcomeresistance towards treatment with STI571. Moreover, knowledge of suchmutations is also very useful in the diagnosis of Ph+ leukemias in thatit allows e.g. the detection of drug-resistant clones before clinicalrelapse of the patient.

[0005] Definitions:

[0006] Within the context of this disclosure the following expressions,terms and abbreviations have the meanings as defined below:

[0007] In the expression “a mutated functional Abl kinase domain”, thepart “mutated Abl kinase domain” refers to the native human Abl kinasedomain containing mutations including amino acid exchanges, amino aciddeletions and/or amino acid additions.

[0008] In the expression “a mutated functional Abl kinase domain”, theterm “functional” indicates that the respective kinase domain possessestyrosine kinase activity. Preferably, the kinase activity of the mutatedfunctional Abl kinase domain is in the range of that of the native humanAbl kinase domain.

[0009] In the expression “a mutated functional Abl kinase domain beingresistant to inhibition of its tyrosine kinase activity by STI571 or asalt thereof”, the term “resistant” means that STI571 inhibits therespective mutated functional Abl kinase domain with an IC₅₀ that ishigher than that of the native human Abl kinase domain, i.e. higher thanabout 0.025 μM, preferably higher than about 0.15 μM, more preferablyhigher than about 0.25 μM, most preferably higher than about 5 μM.

[0010] In the expression “amino acid sequence of the native human Ablkinase domain or an essentially similar sequence thereof”, the part “oran essentially similar sequence thereof” refers to the amino acidsequence of the native human Abl kinase domain containing mutations,including amino acid exchanges, amino acid deletions and/or amino acidadditions, that are not essential for the functionality of the kinaseand its resistance to inhibition by STI571 or a salt thereof within themeaning of the term “functional” and “resistant” as defined hereinabove.

[0011] The expression “replaced by another amino acid” refers to thereplacement of a certain natural amino acid by another natural aminoacid.

[0012] The names of the amino acids are either written out or the oneletter or three letter codes are used. Mutations are referred to byaccepted nomenclature, e.g. “Ala380Thr” or “380 Ala→Thr” both indicatingthat alanine at position 380 is replaced by threonine.

[0013] SEQ ID NO: 1 represents the cDNA coding for the native human Ablprotein (human c-abl mRNA; GenBank Accession No.: X16416).

[0014] SEQ ID NO: 2 represents the amino acid sequence of the nativehuman Abl protein (human c-Abl; SwissProt Acc. No.: P00519).

[0015] Unless indicated otherwise, the number given for a certain aminoacid refers to the numbering of the amino acids in SEQ ID NO: 2. In anamino acid sequence that is essentially similar to the amino acidsequence of the native human Abl kinase domain within the meaning asdefined above, the amino acids are numbered in accordance with thenumbering of the amino acids in SEQ ID NO: 2.

[0016] The term “isolated” means that the material is removed from itsoriginal environment (e.g., the natural environment if it is naturallyoccurring).

[0017] A “host cell”, refers to a prokaryotic or eukaryotic cell thatcontains heterologous DNA that has been introduced into the cell by anymeans, e.g., electroporation, calcium phosphate precipitation,microinjection, transformation, viral infection, and the like.

DESCRIPTION OF THE INVENTION

[0018] In practicing the present invention, many conventional techniquesin molecular biology, microbiology, and recombinant DNA are used. Thesetechniques are well known and are explained in, for example, CurrentProtocols in Molecular Biology, Volumes I, II, and III, 1997 (F. M.Ausubel ed.); Sambrook et al., 1989, Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.; DNA Cloning: A Practical Approach, Volumes I and II, 1985(D. N. Glover ed.); Oligonucleotide Synthesis, 1984 (M. L. Gait ed.);Nucleic Acid Hybridization, 1985, (Hames and Higgins); Transcription andTranslation, 1984 (Hames and Higgins eds.); Animal Cell Culture, 1986(R. I. Freshney ed.); Immobilized Cells and Enzymes, 1986 (IRL Press);Perbal, 1984, A Practical Guide to Molecular Cloning; the series,Methods in Enzymology (Academic Press, Inc.); Gene Transfer Vectors forMammalian Cells, 1987 (J. H. Miller and M. P. Calos eds., Cold SpringHarbor Laboratory); and Methods in Enzymology Vol. 154 and Vol. 155 (Wuand Grossman, and Wu, eds., respectively).

[0019] In particular, the polypeptides of the present invention can beproduced by recombinant DNA technology using techniques well-known inthe art. Methods which are well known to those skilled in the art can beused to construct expression vectors containing the sequences encodingthe polypeptides of the invention and appropriatetranscriptional/translational control signals. A variety ofhost-expression vector systems can be utilized to express thepolypeptides of the invention.

[0020] (1) The invention relates to an isolated polypeptide whichcomprises a mutated functional Abl kinase domain that is resistant toinhibition of its tyrosine kinase activity byN-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-piperazin-1-ylmethyl)-benzamideor a salt thereof.

[0021] (2) The invention further relates in particular to an isolatedpolypeptide which comprises a mutated functional Abl kinase domaincomprising the amino acid sequence of the native human Abl kinase domainor an essentially similar sequence thereof in which at least one aminoacid is replaced by another amino acid, said mutated functional Ablkinase domain being resistant to inhibition of its tyrosine kinaseactivity byN-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-piperazin-1-ylmethyl)-benzamideor a salt thereof.

[0022] (3) The invention especially relates to an isolated polypeptidewhich comprises a mutated functional Abl kinase domain comprising theamino acid sequence of the native human Abl kinase domain or anessentially similar sequence thereof in which at least one amino acidselected from Leu248, Glu255, Lys271, Glu286, Met290, Thr315, Tyr320,Asn322, Glu373, His375 and Ala380 is replaced by another amino acid,said mutated functional Abl kinase domain being resistant to inhibitionof its tyrosine kinase activity byN-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-piperazin-1-ylmethyl)-benzamide or a salt thereof.

[0023] (4) A preferred embodiment of the invention relates to anisolated polypeptide which comprises a mutated functional Abl kinasedomain comprising the amino acid sequence of the native human Abl kinasedomain or an essentially similar sequence thereof in which at least oneamino acid selected from Leu248, Glu255, Lys271, Glu286, Met290, Tyr320,Asn322, Glu373, His375 and Ala380 is replaced by another amino acid,said mutated functional Abl kinase domain being resistant to inhibitionof its tyrosine kinase activity byN-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-piperazin-1-ylmethyl)-benzamide or a salt thereof.

[0024] (5) Another preferred embodiment of the invention relates to anisolated polypeptide which comprises a mutated functional Abl kinasedomain comprising the amino acid sequence of the native human Abl kinasedomain or an essentially similar sequence thereof in which at least oneamino acid selected from Leu248, Lys271, Glu286, Met290, Tyr320, Asn322,Glu373, His375 and Ala380 is replaced by another amino acid, saidmutated functional Abl kinase domain being resistant to inhibition ofits tyrosine kinase activity byN-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-piperazin-1-ylmethyl)-benzamide or a salt thereof.

[0025] (6) Another especially preferred embodiment of the inventionrelates to an isolated polypeptide which comprises a mutated functionalAbl kinase domain comprising the amino acid sequence of the native humanAbl kinase domain or an essentially similar sequence thereof in which atleast one amino acid selected from Glu255, Thr315 and Ala380 is replacedby another amino acid, said mutated functional Abl kinase domain beingresistant to inhibition of its tyrosine kinase activity byN-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-piperazin-1-ylmethyl)-benzamideor a salt thereof.

[0026] (7) Another very preferred embodiment of the invention relates toan isolated polypeptide which comprises a mutated functional Abl kinasedomain comprising the amino acid sequence of the native human Abl kinasedomain or an essentially similar sequence thereof in which at least oneamino acid selected from Glu255 and Ala380 is replaced by another aminoacid, said mutated functional Abl kinase domain being resistant toinhibition of its tyrosine kinase activity byN-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-piperazin-1-ylmethyl)-benzamideor a salt thereof.

[0027] (8) Most preferably the invention relates to an isolatedpolypeptide according to any one of the preceding paragraphs (2)-(7),wherein in the amino acid sequence of the native human Abl kinase domainor an essentially similar sequence thereof a single amino acid isreplaced by another amino acid.

[0028] (9) The invention relates very especially preferred to anisolated polypeptide which comprises a mutated functional Abl kinasedomain comprising the amino acid sequence of the native human Abl kinasedomain or an essentially similar sequence thereof in which Glu255 isreplaced by another amino acid, said mutated functional Abl kinasedomain being resistant to inhibition of its tyrosine kinase activity byN-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-piperazin-1-ylmethyl)-benzamideor a salt thereof.

[0029] (10) Most especially preferred the invention relates to anisolated polypeptide which comprises a mutated functional Abl kinasedomain comprising the amino acid sequence of the native human Abl kinasedomain or an essentially similar sequence thereof that contains at leastone amino acid mutation selected from Glu255Val, Glu255Lys, Thr315Val,Thr315Leu, Thr315Met, Thr315Gln, Thr315Phe and Ala380Thr, said mutatedfunctional Abl kinase domain being resistant to inhibition of itstyrosine kinase activity byN-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-piperazin-1-ylmethyl)-benzamideor a salt thereof.

[0030] (11) In a further very preferred embodiment the invention relatesto an isolated polypeptide which comprises a mutated functional Ablkinase domain comprising the amino acid sequence of the native human Ablkinase domain or an essentially similar sequence thereof that containsat least one amino acid mutation selected from Glu255Val, Thr315Val,Thr315Leu, Thr315Met, Thr315Gln, Thr315Phe and Ala380Thr, said mutatedfunctional Abl kinase domain being resistant to inhibition of itstyrosine kinase activity byN-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-piperazin-1-ylmethyl)-benzamide or a salt thereof.

[0031] (12) In another especially preferred embodiment the inventionrelates to an isolated polypeptide which comprises a mutated functionalAbl kinase domain comprising the amino acid sequence of the native humanAbl kinase domain or an essentially similar sequence thereof thatcontains at least one amino acid mutation selected from Thr315Leu,Thr315Met, Thr315Gln and Thr315Phe, said mutated functional Abl kinasedomain being resistant to inhibition of its tyrosine kinase activity byN-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-piperazin-1-ylmethyl)-benzamideor a salt thereof.

[0032] (13) Most preferably the invention relates to an isolatedpolypeptide according to any one of the preceding paragraphs (10)-(12),wherein the amino acid sequence of the native human Abl kinase domain oran essentially similar sequence thereof contains a single amino acidmutation.

[0033] (14) Preferred above all the invention relates to an isolatedpolypeptide which comprises a mutated functional Abl kinase domaincomprising the amino acid sequence of the native human Abl kinase domainor an essentially similar sequence thereof that contains the amino acidmutation Glu255Val, said mutated functional Abl kinase domain beingresistant to inhibition of its tyrosine kinase activity byN-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-piperazin-1-ylmethyl)-benzamideor a salt thereof.

[0034] (15) In a preferred embodiment the invention relates to anisolated polypeptide according to any one of the preceding pragraphs(2)-(14), wherein the amino acid sequence of the native human Abl kinasedomain consists of amino acids 229-500 of SEQ ID NO: 2.

[0035] (16) In another preferred embodiment the invention relates to anisolated polypeptide according to any one of the preceding paragraphs(2)-(15), said isolated polypeptide being a Bcr-Abl tyrosine kinase.

[0036] (17) In yet another preferred embodiment the invention relates tothe use of an isolated polypeptide of any one of the preceding pragraphs(2) to (16) to screen for compounds which inhibit the tyrosine kinaseactivity of said polypeptide.

[0037] (18) The invention also relates to an isolated nucleic acidmolecule comprising a nucleotide sequence that encodes a polypeptideaccording to any one of the preceding paragraphs (2)-(16).

[0038] (19) The invention further relates to the use of a nucleic acidmolecule of the preceding paragraph (18) in the production of apolypeptide of any one of the preceding paragraphs (2) to (16) for usein screening for compounds which inhibit the tyrosine kinase activity ofsaid polypeptide.

[0039] (20) The invention also relates to a recombinant vectorcomprising a nucleic acid molecule according to the preceding paragraph(18).

[0040] (21) The invention further relates especially to a recombinantvector according to the preceding paragraph (20), which is a recombinantexpression vector.

[0041] (22) The invention also relates to a host cell comprising arecombinant vector according to the preceding paragraph (20) or (21).

[0042] Preferably the invention relates to an isolated polypeptide whichcomprises a mutated functional Abl kinase domain comprising the aminoacid sequence of the native human Abl kinase domain in which at leastone amino acid is replaced by another amino acid, said mutatedfunctional Abl kinase domain being resistant to inhibition of itstyrosine kinase activity byN-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-piperazin-1-ylmethyl)-benzamide or a salt thereof.

[0043] Most preferred are the mutations described herein, which arepresent in patients who suffer from Philadelphia chromosome-positiveleukemia and are resistant against treatment with STI571.

[0044] A preferred salt of STI571 is the methane sulfonate saltdescribed in WO 99/03854.

[0045] Screening for compounds which inhibit the tyrosine kinaseactivity of the polypeptides of the invention may be done for example byusing an isolated polypeptide of the invention in any in vitro tyrosinekinase phosphorylation assay known in the art and determining thepotential of a compound to inhibit the tyrosine kinase activity of apolypeptide of the invention in such an assay.

[0046] High-throughput screening assays known in the art may be used toscreen large compound libraries for compounds which inhibit the tyrosinekinase activity of the polypeptides of the invention.

[0047] Besides the random screening of large compound libraries, thepolypeptides of the present invention may also be used in the followingscreening approach: The 3-dimensional structure of a polypeptide of theinvention is determined by e.g. X-ray crystallography. The atomiccoordinates of a polypeptide of the invention are then used to design apotential inhibitor. Said potential inhibitor is then synthesized andtested for its ability to inhibit the tyrosine kinase activity of thepolypeptide of the invention in any in vitro tyrosine kinasephosphorylation assay.

EXAMPLES

[0048] The following Examples serve to illustrate the invention withoutlimiting its scope.

Example 1

[0049] Methods:

[0050] Plasmids and site directed mutagenesis:

[0051] The hybrid cDNA coding for HckAblSH1 was cloned by amplifying therespective DNA fragments from pUCΔNdel/XbalHck [Warmuth M. et al., J.Biol. Chem. 272, 33260-70 (1997)] and pcDNA3bcr-abl. These fragmentswere ligated blunt end to yield pUCΔNdel/XbalhckablSH1. Because of itsrelatively small size when compared to Bcr-Abl or c-Abl, this construct,hckAblSH1, allowed to introduce point mutations into the kinase domainof Abl by a one step cloning procedure. Point mutations were introducedinto hckablSH1 using the QuickChange site directed mutagenesis protocolfrom Stratagene (La Jolla, Calif.). In order to introduce pointmutations into Bcr-Abl, a Kpnl/Eco47III-subfragment of Bcr-Ablcontaining the sequence coding for Bcr-Abl's kinase domain was clonedinto pUCΔNdel/Xbal engineered by site directed mutagenesis to contain anEco47III site in the polylinker. After introduction of point mutations,this fragment was first recloned into pcDNA3abl. Thereafter, the 5′ partof abl up to the Kpnl site was substituted by Bcr coding sequences usinga Kpnl-fragment from pcDNA3bcr-abl. All mutations were confirmed bysequencing. For expression in Cos7 and 32D cells cDNAs were cloned intopApuro.

[0052] Cell Lines:

[0053] Parental 32D cells as well as 32D cells expressing Bcr-Abl andmutants thereof (32Dp210) were grown in Iscove's modified dulbeccosmedia (IMDM) supplemented with 10% FBS. COS7 cells were cultured inDulbecco's modified eagle medium (DMEM) containing 4,5 g/l glucose) andsupplemented with 10% FBS. All media and FBS were purchased from GibcoLife Technologies, Inc, Karlsruhe, Germany.

[0054] Transfection of Cells:

[0055] Cos7 cells were transfected using Effecten® transfection reagentas to the guidelines of the manufacturer (Quiagen, Hilden, Germany). 32Dcells were transfected by electroporation. Puromycin was used forselection at a concentration of 1 μg/ml.

[0056] Cell Lysis:

[0057] Cos7 cells were lysed as described recently [Warmuth M. et al.,J. Biol. Chem. 272, 33260-70 (1997)]. For lysis, exponentially growing32D cells were harvested and washed twice in cold PBS. For experimentsevaluating the activity profile of STI571, cells were incubated with theindicated concentrations of inhibitor or with DMSO at a density of 5×10⁶cells/ml for 1,5-2 h. 10⁷ cells were lysed in 100 μl of lysis buffercontaining 1% NP-40, 20 mM Tris (pH 8,0), 50 mM NaCl, and 10 mM EDTA, 1mM phenylmethylsulfonylfluorid, 10 μg/ml aprotinin, 10 μg/ml leupeptin,and 2 mM sodium orthovanadate. After resuspension in lysis buffer, cellswere incubated for 25 min on ice. Finally, unsoluble material wasremoved by centrifugation at 15,000 g. Clarified lysates were checkedfor protein concentrations using a BioRad protein assay.

[0058] Immunoprecipitation:

[0059] For immunoprecipitation 150 μl of standardized 32D cell lysatewas diluted by addition of 450 μl of incubation buffer containing 20 mMTris (pH 8,0), 50 mM NaCl, and 10 mM EDTA,1 mMphenylmethylsulfonylfluorid, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and2 mM sodium orthovanadate and incubated with 5 μg of the indicatedantibodies for 2 hours on a overhead rotor at 4° C. Sepharose A beads(Pharmacia Biotech Inc., Freiburg, Germany) were prepared by washingtwice in IP buffer [0,1% NP-40, 20 mM Tris (pH 8,0), 50 mM NaCl, and 10mM EDTA, 1 mM phenylmethylsulfonylfluorid, 10 μg/ml aprotinin, 10 μg/mlleupeptin, and 2 mM sodium orthovanadate] and added to each sample for 2additional hours. Finally, the immunoprepicitates were washed threetimes in IP buffer, subsequently boiled in 2× sample buffer and preparedfor SDS-PAGE.

[0060] Gel Electrophoresis and Immunoblotting:

[0061] Gel electrophoresis and immunoblotting were performed asdescribed [Danhauser-Riedl S. et al., Cancer Res. 56, 3589-96 (1996);Warmuth M. et al., J. Biol. Chem. 272, 33260-70 (1997)] with some minormodifications. Proteins were routinely blotted on nitrocellulosemembranes (Schleicher & Schuell, Dassel, Germany). Membranes wereblocked in 5% milk powder for 1 h. Primary and secondary antibodies werediluted 1:500 to 1:5000 in TBS containing 1% milk powder. Proteins weredetected using the ECL or ECL Plus detection system as recommended bythe manufacturer (Amersham, Braunschweig, Germany).

[0062] Detection of Apoptosis by Flow Cytometry:

[0063] For assessing apoptosis induced by the various kinase inhibitors,cells were incubated with the indicated concentrations of STI571 at adensity of 5×10⁴ per ml. Apoptosis was routinely assessed by measuringthe binding of FITC-conjugated Annexin V to the membranes of apoptosingcells. About 5×10⁴ cells were taken at the indicated time points andwashed once in PBS. Thereafter, cells were resuspended in 195 μl ofAnnexin V binding buffer and 5 μl of Annexin V-FITC (Bender MedSystemsDiagnostics, Vienna, Austria) were added. Cells were mixed and incubatedat room temperature for 10-20 min. Afterwards, cells were pelletedagain, washed once and resuspended in 190 μl of Annexin V bindingbuffer. 10 μl of a 20 μg/ml propidium iodide stock solution were addedand the ratio of Annexin V-positive to negative cells was determined byFACS-analysis using a Coulter EPICS XL 4-color cytometer.

[0064] Results:

[0065] Mutations to either Valine (V), Leucine (L), Isoleucine (I),Methionine (M), Glutamine (Q) or Phenylalanine (F) at position 315 andto either Serine (S), Cysteine (C) or Threonine (T) at position 380 weregenerated in a hybrid kinase, HckAblSH1, consisting of the SH2 and SH3domain of Hck and the SH1/kinase domain of Abl. When expressed in Cos7cells, these hybrid kinases and all mutants at position 315 and 380showed a high spontaneous kinase activity, proving that these positionsare not critical for ATP binding (Table 1). In marked contrast, whentested for inhibition by STI571, no inhibition was seen with up to 10 μMof compound for the mutants T315L, T315I, T315M, T315Q and T315F (Table1), whereas HckAblSH1 wild-type (wt) could be inhibited with similarkinetics by STI571 as were found for Bcr-Abl (IC50 cellular tyrosinephosphorylation (IC50_(CTP)) approx. 0.5 μM). The mutants T315V andA380T retained some partial sensitivity but IC50_(CTP) values were stillhigher than 10 μM. In contrast, the mutants A380S and A380C displayedsensitivity to STI571, which was comparable to HckAblSH1 wt (see Table 1for summary). TABLE 1 Influence of mutations of T315 and A380 ofHckAbISH1 on kinase activity and inhibiton by STI571 kinase activityInhibition by STI571 T315V +++ IC50 > 10 T315L +++ CR T315I +++ CR T315M++++ CR T315Q ++ CR T315F ++ CR A380C +++ NS A380S +++ NS A380T + IC50 >10

[0066] All data based on inhibiton of cellular tyrosine phosphorylationof transiently transfected Cos7 cells determined by Western blotanalysis using the monoclonal α-phosphotyrosine antibody PY99. IC50values were determined using scion image software. Complete remission(CR) was defined as no detectable reduction of cellular tyrosinephosphrylation by 10 μM STI571. NS (normal sensitivity)=inhibtion withsimilar kinetics as HckAblSH1 wt.

[0067] Our data identify positions 315 and 380 as critical gatekeepersfor the binding pocket of STI571, which contribute to define thesensitivity of individual protein kinases towards STI571. For example,the STI571-insensitive receptor tyrosine kinase Flt-3, which has highhomology to the c-Kit and the PDGF-R kinases, has a phenylalanine at theposition homologous to T315, which would, based on our data, not be inaccordance with STI571 binding. In a similar way, the resistance of mostother kinases tested with STI571 could be explained.

[0068] In order to investigate whether and to what degree some of theabove described point mutations of the gatekeeper position T315 are ableto induce biological resistance towards STI571 we introduced into fulllength Bcr-Abl the mutations T315V, T315L, T315I, T315M, T315Q andT315F. When expressed in Cos7 cells all mutants displayed kinaseactivity close to or similar to wild-type Bcr-Abl (Bcr-ABLwt). If testedfor inhibition by STI571, identical results were obtained as describedfor the corresponding mutations in HckAblSH1 (Table 2). Similar toBcr-Ablwt, expression of these mutants in 32D, an IL-3-dependent,hematopoietic cell line of murine origin, gave rise to cell linesgrowing IL-3 independently. Exposure of 32DBcr-Ablwt cells to 1 or 10 μMSTI571 lead to a rapid stop of proliferation and induction of apoptoticcell death in more than 90% of cells. On the contrary, if T315 mutantBcr-Abl kinases, for example T315I, were expressed the block inproliferation and the induction of apoptosis caused by 1 μM STI571 werecompletely abolished (Table 2) and the effects of STI571 seen at 10 μMwere reduced to levels found in control experiments using parental 32Dcells grown in the presence of IL-3. Phosphotyrosine blots of samples ofcells expressing either wt or mutant Bcr-Abl proteins confirmed thatmutations at position 315 completely abolished the effect of STI571 onAbl auto- and substrate phosphorylation, with the exception of T315Vwhich was still to some degree inhibited by STI571 but displayed asimilar biological phenotype as the other mutants (Table 2). Thissuggests that the reminder biological activity of STI571 at 10 μM wasrather due to cytotoxicity than to a reminder sersitivity of the mutantsor cross-reaction of STI571 with another tyrosine kinase. In summary,all mutations lifted the IC50 for inhibition of proliferation(IC50_(IOP)) from 0,09 to approximately 7.5 μM and for inhibition ofsurvival (IC50_(IOS)) from 0,5 to more than 10 μM (Table 2). Takentogether, these data show that mutations of “molecular gatekeeper”positions as described above are able to confer complete biologicalresistance towards STI571 in a cell culture model. TABLE 2 Biochemicaland biological characterization of mutations of T315 in Bcr-Abl to aminoacids with longer side chains IC50 (μM) cellular Induction of factor-IC50 (μM) IC50 (μM) kinase tyrosine phosphorylation independent growthapoptosis proliferation mutant activity Cos7 32D in 32D 32D 32D(32D) >10 7.5 wt +++ 0.25 0.25 yes 0.5 0.09 T315V +++ >10 >10 yes >107.5 T315L ++++ c.r. c.r. yes >10 7.5 T315I +++ c.r. c.r. yes >10 7.5T315M ++++ c.r. c.r. yes >10 7.5 T315Q +++ c.r. c.r. yes >10 7.5 T315F+++ c.r. c.r. yes >10 7.5

Example 2

[0069] STI571 inhibits the Abl tyrosine kinase with an IC₅₀ of 0.025 μMfor purified Bcr-Abl and c-Abl but not the fms or the Src familykinases. The mechanism of inhibition is through competitive inhibitionof ATP binding. To better understand the mechanism of specificity of thetyrosine kinase inhibitor the Abl kinase was compared to a model of theLck kinase domain. This model predicts the following sites are criticalfor STI571 association: L248, Y320, N322, E373, H375 and A380. Each ofthese residues were changed to the corresponding residue in Src or fmsand IC₅₀ values for STI571 with each mutant were determined. L248A andH375L yielded kinase inactive mutants, Y320K, N322S, E373N and A380G hadIC₅₀ values identical to wild type Abl. A380T, however, demonstrated anIC₅₀ of 0.34 μM suggesting that STI571 bound less efficiently when alarger residue replaced the alanine. Recent crystallization of the Ablkinase domain with a related inhibitor shows that the configuration ofthe activation loop of the Abl kinase domain differs significantly fromthat of the Src family kinases. This structure identifies K271, E286,M290, T315, M318 and D381 as critical contacts of STI571. All of theseresidues are conserved between Src and Abl. The last two of these bindSTI571 via their peptide backbone, thus mutants in these residues cannotbe created. The remainder of the residues were mutated to residueslacking the potential for hydrogen bonding and IC₅₀ values weredetermined. K271R, E286L and M290A were kinase inactive. T315V had anIC₅₀ value of 0.35 μM, which is consistent with the crystal structure ofthe Abl kinase domain which predicts that the side chain of T315 forms acritical hydrogen bond with STI571.

Example 3

[0070] A group of 32 patients who are either refractory to treatmentwith STI571 or who relapsed whilst being treated were investigated. Themedian duration of therapy was 95 days; prior to STI571 treatment, twopatients were in chronic phase, nine in accelerated phase, 20 in myeloidand, and one in lymphoid blast crisis of the disease. Reversetranscriptase-polymerase chain reaction (RT-PCR) products specific forthe Bcr-Abl tyrosine kinase domain were sequenced (Heminested RT-PCR wasperformed to amplify the sequence specifically coding for the Bcr-Abltyrosine kinase: 1^(st) step B2B ACAGAATTCCGCTGACCATCAATAAG andA7−AGACGTCGGACTTGATGGAGAACT; 2^(nd) step FA4+AAGCGCAACAAGCCCACTGTCTATand A7−). An acquired A→T point mutation at position 58802 (GeneBankaccession number U07563, locus HSABLGR3)—which results in a Glu255Valsubstitution—was detected in one patient. Restriction analysis of cDNAand genomic DNA (RT-PCR and genomic PCR were performed using primersA4+TCACCACGCTCCATTATCCA, A4−CTTCCACACGCTCTCGTACA; Mnl I restrictiondigest of PCR products; removal of an Mnl I restriction site as theresult of the point mutation A58802T) was used to confirm the presenceof the mutation and to track it during the course of treatment. Onlywild-type Abl sequence was present before the STI571 therapy. Thepatient was treated with STI571 in late chronic phase, went intocomplete hematologic remission, but progressed to blast crisis afterfive months. Reactivation of Bcr-Abl was confirmed by Crklimmunoblotting [K. Senechal, Mol. Cell. Biol. 18, 5082 (1998)]. Therelative proportion of phosphorylated Crkl (reflecting active Bcr-Abl)was 49% before STI571 therapy, 24% at day 27, 28% at day 83, and 77% atthe time of clinical resistance at day 166. The biological significanceof the Glu255Val change is determined by an Abl autophosphorylationassay. STI571 inhibits wild-type Abl with an IC₅₀ of 0.025 μM. Themutation leads to a virtual insensitivity to STI571, with an IC₅₀ of >5μM.

Example 4

[0071] The Bcr-Abl kinase domain from cells obtained from 12 CML and Ph+acute leukemia patients who relapsed while receiving STI571 wassequenced. A functional point-mutation in the kinase domain in one casewas identified. This was a G→A change that results in a Glu→Lyssubstitution at position 255 of Abl.

Example 5

[0072] Patients and Sample Preparation:

[0073] Thirty bone marrow samples from 21 patients with Ph+ALL who wereenrolled into consecutive “Phase II study to determine the safety andanti-leukemic effect of STI571 in adult patients with Ph+ acuteleukemias” were analyzed. According to the study protocol, thesepatients had relapsed ALL or were refractory after at least 2 cycles ofstandard chemotherapy. From all of the patients, samples were obtainedbefore beginning STI571 treatment: 13 of these samples were fromindividuals who later were classified as good responders to STI571 (Nos.1-13, sensitive, S) including 12 patients with hematological completeremission (CR) and one patient with partial remission (PR) but completeperipheral hematological recovery (No. 1). Eight samples were collectedfrom individuals who later were found not to respond to STI571 (Nos.14-21, primarily resistant, R) including 6 patients without anyhematological response, one with cytoreduction in the bone marrow butpersistent peripheral leukemic cells (No. 20) and another with PR butincomplete peripheral hematological recovery (No. 16). Matched bonemarrow samples from 9 patients (Nos. 1-5 and Nos. 14-17) were alsoobtained while they were on treatment with STI571. Mononuclear cellswere separated by density gradient centrifugation through Ficoll-Hypaque(Biochrom, Berlin, Germany). Total RNA was extracted using the acidguanidium/phenol/chloroform method with minor modifications [Puissant C.and Houdebine L. M., Biotechniques 8, 148-149 (1990)]. Only samples withleukemic blast cell infiltration of more than 80% were included into theanalysis.

[0074] Reverse Transcription Polymerase Chain Reaction and SequencingAnalysis:

[0075] One microgram of total RNA was used for reverse transcription(RT) by Superscript II RT (Life Technologies, Grand Island, N.Y.)according to standard protocols. Primers specific for the ATP bindingsite of ABL including the “loop” were designed using gene bankinformation GI6382056: ATP-F 5′-GCG CAA CAA GCC CAC TGT CT-3′; ATP-R5′-GCA CTC CCT CAG GTA GTC CA-3′ and LOOP-F 5′-TGG ACT ACC TGA GGG AGTGC-3′; LOOP-R 5′-CGG TAG TCC TTC TCT AGC AGC-3′. Oligonucleotides weresynthesized by Life Technologies. Polymerase chain reaction (PCR) wasperformed as described previously [Hofmann W. K. et al., Leuk. Res. 25,333-338 (2001)] using an annealing temperature of 58° C. PCR-productswere separated on a 2% agarose gel containing 0.3 mg/ml ethidium bromideand purified using the QIAquick purification system (Qiagen, Valencia,Calif.) according to the manufacturers protocol. The purified DNA wasdirectly sequenced in both directions (sense and antisense) by the ABIPRISM dye terminator cycle sequencing reaction (Perkin-Elmer, Foster,Calif.).

[0076] Results:

[0077] Analysis of the sequence of the ATP binding site revealed asingle point mutation at nucleotide 1127 (GI6382056) changing a G to anA resulting in a substitution at codon 255 of Lys (mutant) for a Glu(wild-type). This mutation was found in 6 samples from patients afterthey were treated with STI571 (Nos. 1, 2, 4, 5, 15, 16) but mutationswere not found in any other sample including the matched samples fromthe patients before beginning treatment with STI571 (Table 3). Thechange was verified by sequencing from both the sense and antisensedirections. In addition, one sample (No. 17) from a patient with anaberrant cALL had a single point mutation at nucleotide 1308 changing aC to T resulting in a substitution at codon 315 of isoleucine (mutant)for a threonine (wild-type). This sample was unusual because the cellsalso expressed CD33, a cell surface protein expressed on myeloid cells.

[0078] Our data strongly suggest that E255K developed during treatmentwith STI571. Our analysis of matched samples found, that none of thesamples from untreated patients (including sensitive patients and thosewith primary resistance) had this mutation. In contrast, six of 9samples (67%) from these patients undergoing treatment with STI571 hadthis substitution at E255. The overall frequency of mutations in the ATPbinding site was 7 of 9 (78%) in our paired bone marrow samples frompatients undergoing therapy with STI571. TABLE 3 atched bone marrowsamples: Development of mutations in the egion coding for the ATPbinding site of ABL during treatment of h+ ALL with STI571. ABS statusprior to Response ABS status after No. Diagnosis treatment with STI571to STI571 treatment with STI571 1 Ph+ cALL Wild type PR E255K 2 Ph+ cALLWild type CR E255K 3 Ph+ cALL Wild type CR Wild type 4 Ph+ cALL Wildtype CR E255K 5 Ph+ cALL Wild type CR E255K 14  Ph+ cALL Wild type noWild type 15  Ph+ cALL Wild type no E255K 16  Ph+ pre B-ALL Wild type PRE255K 17  Ph+ cALL, CD33+ Wild type no T315I

[0079]

1 2 1 3393 DNA Homo sapiens CDS (1)..(3393) 1 atg ttg gag atc tgc ctgaag ctg gtg ggc tgc aaa tcc aag aag ggg 48 Met Leu Glu Ile Cys Leu LysLeu Val Gly Cys Lys Ser Lys Lys Gly 1 5 10 15 ctg tcc tcg tcc tcc agctgt tat ctg gaa gaa gcc ctt cag cgg cca 96 Leu Ser Ser Ser Ser Ser CysTyr Leu Glu Glu Ala Leu Gln Arg Pro 20 25 30 gta gca tct gac ttt gag cctcag ggt ctg agt gaa gcc gct cgt tgg 144 Val Ala Ser Asp Phe Glu Pro GlnGly Leu Ser Glu Ala Ala Arg Trp 35 40 45 aac tcc aag gaa aac ctt ctc gctgga ccc agt gaa aat gac ccc aac 192 Asn Ser Lys Glu Asn Leu Leu Ala GlyPro Ser Glu Asn Asp Pro Asn 50 55 60 ctt ttc gtt gca ctg tat gat ttt gtggcc agt gga gat aac act cta 240 Leu Phe Val Ala Leu Tyr Asp Phe Val AlaSer Gly Asp Asn Thr Leu 65 70 75 80 agc ata act aaa ggt gaa aag ctc cgggtc tta ggc tat aat cac aat 288 Ser Ile Thr Lys Gly Glu Lys Leu Arg ValLeu Gly Tyr Asn His Asn 85 90 95 ggg gaa tgg tgt gaa gcc caa acc aaa aatggc caa ggc tgg gtc cca 336 Gly Glu Trp Cys Glu Ala Gln Thr Lys Asn GlyGln Gly Trp Val Pro 100 105 110 agc aac tac atc acg cca gtc aac agt ctggag aaa cac tcc tgg tac 384 Ser Asn Tyr Ile Thr Pro Val Asn Ser Leu GluLys His Ser Trp Tyr 115 120 125 cat ggg cct gtg tcc cgc aat gcc gct gagtat ctg ctg agc agc ggg 432 His Gly Pro Val Ser Arg Asn Ala Ala Glu TyrLeu Leu Ser Ser Gly 130 135 140 atc aat ggc agc ttc ttg gtg cgt gag agtgag agc agt cct ggc cag 480 Ile Asn Gly Ser Phe Leu Val Arg Glu Ser GluSer Ser Pro Gly Gln 145 150 155 160 agg tcc atc tcg ctg aga tac gaa gggagg gtg tac cat tac agg atc 528 Arg Ser Ile Ser Leu Arg Tyr Glu Gly ArgVal Tyr His Tyr Arg Ile 165 170 175 aac act gct tct gat ggc aag ctc tacgtc tcc tcc gag agc cgc ttc 576 Asn Thr Ala Ser Asp Gly Lys Leu Tyr ValSer Ser Glu Ser Arg Phe 180 185 190 aac acc ctg gcc gag ttg gtt cat catcat tca acg gtg gcc gac ggg 624 Asn Thr Leu Ala Glu Leu Val His His HisSer Thr Val Ala Asp Gly 195 200 205 ctc atc acc acg ctc cat tat cca gcccca aag cgc aac aag ccc act 672 Leu Ile Thr Thr Leu His Tyr Pro Ala ProLys Arg Asn Lys Pro Thr 210 215 220 gtc tat ggt gtg tcc ccc aac tac gacaag tgg gag atg gaa cgc acg 720 Val Tyr Gly Val Ser Pro Asn Tyr Asp LysTrp Glu Met Glu Arg Thr 225 230 235 240 gac atc acc atg aag cac aag ctgggc ggg ggc cag tac ggg gag gtg 768 Asp Ile Thr Met Lys His Lys Leu GlyGly Gly Gln Tyr Gly Glu Val 245 250 255 tac gag ggc gtg tgg aag aaa tacagc ctg acg gtg gcc gtg aag acc 816 Tyr Glu Gly Val Trp Lys Lys Tyr SerLeu Thr Val Ala Val Lys Thr 260 265 270 ttg aag gag gac acc atg gag gtggaa gag ttc ttg aaa gaa gct gca 864 Leu Lys Glu Asp Thr Met Glu Val GluGlu Phe Leu Lys Glu Ala Ala 275 280 285 gtc atg aaa gag atc aaa cac cctaac ctg gtg cag ctc ctt ggg gtc 912 Val Met Lys Glu Ile Lys His Pro AsnLeu Val Gln Leu Leu Gly Val 290 295 300 tgc acc cgg gag ccc ccg ttc tatatc atc act gag ttc atg acc tac 960 Cys Thr Arg Glu Pro Pro Phe Tyr IleIle Thr Glu Phe Met Thr Tyr 305 310 315 320 ggg aac ctc ctg gac tac ctgagg gag tgc aac cgg cag gag gtg aac 1008 Gly Asn Leu Leu Asp Tyr Leu ArgGlu Cys Asn Arg Gln Glu Val Asn 325 330 335 gcc gtg gtg ctg ctg tac atggcc act cag atc tcg tca gcc atg gag 1056 Ala Val Val Leu Leu Tyr Met AlaThr Gln Ile Ser Ser Ala Met Glu 340 345 350 tac ctg gag aag aaa aac ttcatc cac aga gat ctt gct gcc cga aac 1104 Tyr Leu Glu Lys Lys Asn Phe IleHis Arg Asp Leu Ala Ala Arg Asn 355 360 365 tgc ctg gta ggg gag aac cacttg gtg aag gta gct gat ttt ggc ctg 1152 Cys Leu Val Gly Glu Asn His LeuVal Lys Val Ala Asp Phe Gly Leu 370 375 380 agc agg ttg atg aca ggg gacacc tac aca gcc cat gct gga gcc aag 1200 Ser Arg Leu Met Thr Gly Asp ThrTyr Thr Ala His Ala Gly Ala Lys 385 390 395 400 ttc ccc atc aaa tgg actgca ccc gag agc ctg gcc tac aac aag ttc 1248 Phe Pro Ile Lys Trp Thr AlaPro Glu Ser Leu Ala Tyr Asn Lys Phe 405 410 415 tcc atc aag tcc gac gtctgg gca ttt gga gta ttg ctt tgg gaa att 1296 Ser Ile Lys Ser Asp Val TrpAla Phe Gly Val Leu Leu Trp Glu Ile 420 425 430 gct acc tat ggc atg tcccct tac ccg gga att gac ctg tcc cag gtg 1344 Ala Thr Tyr Gly Met Ser ProTyr Pro Gly Ile Asp Leu Ser Gln Val 435 440 445 tat gag ctg cta gag aaggac tac cgc atg gag cgc cca gaa ggc tgc 1392 Tyr Glu Leu Leu Glu Lys AspTyr Arg Met Glu Arg Pro Glu Gly Cys 450 455 460 cca gag aag gtc tat gaactc atg cga gca tgt tgg cag tgg aat ccc 1440 Pro Glu Lys Val Tyr Glu LeuMet Arg Ala Cys Trp Gln Trp Asn Pro 465 470 475 480 tct gac cgg ccc tccttt gct gaa atc cac caa gcc ttt gaa aca atg 1488 Ser Asp Arg Pro Ser PheAla Glu Ile His Gln Ala Phe Glu Thr Met 485 490 495 ttc cag gaa tcc agtatc tca gac gaa gtg gaa aag gag ctg ggg aaa 1536 Phe Gln Glu Ser Ser IleSer Asp Glu Val Glu Lys Glu Leu Gly Lys 500 505 510 caa ggc gtc cgt ggggct gtg agt acc ttg ctg cag gcc cca gag ctg 1584 Gln Gly Val Arg Gly AlaVal Ser Thr Leu Leu Gln Ala Pro Glu Leu 515 520 525 ccc acc aag acg aggacc tcc agg aga gct gca gag cac aga gac acc 1632 Pro Thr Lys Thr Arg ThrSer Arg Arg Ala Ala Glu His Arg Asp Thr 530 535 540 act gac gtg cct gagatg cct cac tcc aag ggc cag gga gag agc gat 1680 Thr Asp Val Pro Glu MetPro His Ser Lys Gly Gln Gly Glu Ser Asp 545 550 555 560 cct ctg gac catgag cct gcc gtg tct cca ttg ctc cct cga aaa gag 1728 Pro Leu Asp His GluPro Ala Val Ser Pro Leu Leu Pro Arg Lys Glu 565 570 575 cga ggt ccc ccggag ggc ggc ctg aat gaa gat gag cgc ctt ctc ccc 1776 Arg Gly Pro Pro GluGly Gly Leu Asn Glu Asp Glu Arg Leu Leu Pro 580 585 590 aaa gac aaa aagacc aac ttg ttc agc gcc ttg atc aag aag aag aag 1824 Lys Asp Lys Lys ThrAsn Leu Phe Ser Ala Leu Ile Lys Lys Lys Lys 595 600 605 aag aca gcc ccaacc cct ccc aaa cgc agc agc tcc ttc cgg gag atg 1872 Lys Thr Ala Pro ThrPro Pro Lys Arg Ser Ser Ser Phe Arg Glu Met 610 615 620 gac ggc cag ccggag cgc aga ggg gcc ggc gag gaa gag ggc cga gac 1920 Asp Gly Gln Pro GluArg Arg Gly Ala Gly Glu Glu Glu Gly Arg Asp 625 630 635 640 atc agc aacggg gca ctg gct ttc acc ccc ttg gac aca gct gac cca 1968 Ile Ser Asn GlyAla Leu Ala Phe Thr Pro Leu Asp Thr Ala Asp Pro 645 650 655 gcc aag tcccca aag ccc agc aat ggg gct ggg gtc ccc aat gga gcc 2016 Ala Lys Ser ProLys Pro Ser Asn Gly Ala Gly Val Pro Asn Gly Ala 660 665 670 ctc cgg gagtcc ggg ggc tca ggc ttc cgg tct ccc cac ctg tgg aag 2064 Leu Arg Glu SerGly Gly Ser Gly Phe Arg Ser Pro His Leu Trp Lys 675 680 685 aag tcc agcacg ctg acc agc agc cgc cta gcc acc ggc gag gag gag 2112 Lys Ser Ser ThrLeu Thr Ser Ser Arg Leu Ala Thr Gly Glu Glu Glu 690 695 700 ggc ggt ggcagc tcc agc aag cgc ttc ctg cgc tct tgc tcc gcc tcc 2160 Gly Gly Gly SerSer Ser Lys Arg Phe Leu Arg Ser Cys Ser Ala Ser 705 710 715 720 tgc gttccc cat ggg gcc aag gac acg gag tgg agg tca gtc acg ctg 2208 Cys Val ProHis Gly Ala Lys Asp Thr Glu Trp Arg Ser Val Thr Leu 725 730 735 cct cgggac ttg cag tcc acg gga aga cag ttt gac tcg tcc aca ttt 2256 Pro Arg AspLeu Gln Ser Thr Gly Arg Gln Phe Asp Ser Ser Thr Phe 740 745 750 gga gggcac aaa agt gag aag ccg gct ctg cct cgg aag agg gca ggg 2304 Gly Gly HisLys Ser Glu Lys Pro Ala Leu Pro Arg Lys Arg Ala Gly 755 760 765 gag aacagg tct gac cag gtg acc cga ggc aca gta acg cct ccc ccc 2352 Glu Asn ArgSer Asp Gln Val Thr Arg Gly Thr Val Thr Pro Pro Pro 770 775 780 agg ctggtg aaa aag aat gag gaa gct gct gat gag gtc ttc aaa gac 2400 Arg Leu ValLys Lys Asn Glu Glu Ala Ala Asp Glu Val Phe Lys Asp 785 790 795 800 atcatg gag tcc agc ccg ggc tcc agc ccg ccc aac ctg act cca aaa 2448 Ile MetGlu Ser Ser Pro Gly Ser Ser Pro Pro Asn Leu Thr Pro Lys 805 810 815 cccctc cgg cgg cag gtc acc gtg gcc cct gcc tcg ggc ctc ccc cac 2496 Pro LeuArg Arg Gln Val Thr Val Ala Pro Ala Ser Gly Leu Pro His 820 825 830 aaggaa gaa gct gaa aag ggc agt gcc tta ggg acc cct gct gca gct 2544 Lys GluGlu Ala Glu Lys Gly Ser Ala Leu Gly Thr Pro Ala Ala Ala 835 840 845 gagcca gtg acc ccc acc agc aaa gca ggc tca ggt gca cca ggg ggc 2592 Glu ProVal Thr Pro Thr Ser Lys Ala Gly Ser Gly Ala Pro Gly Gly 850 855 860 accagc aag ggc ccc gcc gag gag tcc aga gtg agg agg cac aag cac 2640 Thr SerLys Gly Pro Ala Glu Glu Ser Arg Val Arg Arg His Lys His 865 870 875 880tcc tct gag tcg cca ggg agg gac aag ggg aaa ttg tcc agg ctc aaa 2688 SerSer Glu Ser Pro Gly Arg Asp Lys Gly Lys Leu Ser Arg Leu Lys 885 890 895cct gcc ccg ccg ccc cca cca gca gcc tct gca ggg aag gct gga gga 2736 ProAla Pro Pro Pro Pro Pro Ala Ala Ser Ala Gly Lys Ala Gly Gly 900 905 910aag ccc tcg cag agc ccg agc cag gag gcg gcc ggg gag gca gtc ctg 2784 LysPro Ser Gln Ser Pro Ser Gln Glu Ala Ala Gly Glu Ala Val Leu 915 920 925ggc gca aag aca aaa gcc acg agt ctg gtt gat gct gtg aac agt gac 2832 GlyAla Lys Thr Lys Ala Thr Ser Leu Val Asp Ala Val Asn Ser Asp 930 935 940gct gcc aag ccc agc cag ccg gga gag ggc ctc aaa aag ccc gtg ctc 2880 AlaAla Lys Pro Ser Gln Pro Gly Glu Gly Leu Lys Lys Pro Val Leu 945 950 955960 ccg gcc act cca aag cca cag tcc gcc aag ccg tcg ggg acc ccc atc 2928Pro Ala Thr Pro Lys Pro Gln Ser Ala Lys Pro Ser Gly Thr Pro Ile 965 970975 agc cca gcc ccc gtt ccc tcc acg ttg cca tca gca tcc tcg gcc ctg 2976Ser Pro Ala Pro Val Pro Ser Thr Leu Pro Ser Ala Ser Ser Ala Leu 980 985990 gca ggg gac cag ccg tct tcc act gcc ttc atc cct ctc ata tca acc 3024Ala Gly Asp Gln Pro Ser Ser Thr Ala Phe Ile Pro Leu Ile Ser Thr 995 10001005 cga gtg tct ctt cgg aaa acc cgc cag cct cca gag cgg atc gcc 3069Arg Val Ser Leu Arg Lys Thr Arg Gln Pro Pro Glu Arg Ile Ala 1010 10151020 agc ggc gcc atc acc aag ggc gtg gtc ctg gac agc acc gag gcg 3114Ser Gly Ala Ile Thr Lys Gly Val Val Leu Asp Ser Thr Glu Ala 1025 10301035 ctg tgc ctc gcc atc tct agg aac tcc gag cag atg gcc agc cac 3159Leu Cys Leu Ala Ile Ser Arg Asn Ser Glu Gln Met Ala Ser His 1040 10451050 agc gca gtg ctg gag gcc ggc aaa aac ctc tac acg ttc tgc gtg 3204Ser Ala Val Leu Glu Ala Gly Lys Asn Leu Tyr Thr Phe Cys Val 1055 10601065 agc tat gtg gat tcc atc cag caa atg agg aac aag ttt gcc ttc 3249Ser Tyr Val Asp Ser Ile Gln Gln Met Arg Asn Lys Phe Ala Phe 1070 10751080 cga gag gcc atc aac aaa ctg gag aat aat ctc cgg gag ctt cag 3294Arg Glu Ala Ile Asn Lys Leu Glu Asn Asn Leu Arg Glu Leu Gln 1085 10901095 atc tgc ccg gcg aca gca ggc agt ggt ccg gcg gcc act cag gac 3339Ile Cys Pro Ala Thr Ala Gly Ser Gly Pro Ala Ala Thr Gln Asp 1100 11051110 ttc agc aag ctc ctc agt tcg gtg aag gaa atc agt gac ata gtg 3384Phe Ser Lys Leu Leu Ser Ser Val Lys Glu Ile Ser Asp Ile Val 1115 11201125 cag agg tag 3393 Gln Arg 1130 2 1130 PRT Homo sapiens 2 Met Leu GluIle Cys Leu Lys Leu Val Gly Cys Lys Ser Lys Lys Gly 1 5 10 15 Leu SerSer Ser Ser Ser Cys Tyr Leu Glu Glu Ala Leu Gln Arg Pro 20 25 30 Val AlaSer Asp Phe Glu Pro Gln Gly Leu Ser Glu Ala Ala Arg Trp 35 40 45 Asn SerLys Glu Asn Leu Leu Ala Gly Pro Ser Glu Asn Asp Pro Asn 50 55 60 Leu PheVal Ala Leu Tyr Asp Phe Val Ala Ser Gly Asp Asn Thr Leu 65 70 75 80 SerIle Thr Lys Gly Glu Lys Leu Arg Val Leu Gly Tyr Asn His Asn 85 90 95 GlyGlu Trp Cys Glu Ala Gln Thr Lys Asn Gly Gln Gly Trp Val Pro 100 105 110Ser Asn Tyr Ile Thr Pro Val Asn Ser Leu Glu Lys His Ser Trp Tyr 115 120125 His Gly Pro Val Ser Arg Asn Ala Ala Glu Tyr Leu Leu Ser Ser Gly 130135 140 Ile Asn Gly Ser Phe Leu Val Arg Glu Ser Glu Ser Ser Pro Gly Gln145 150 155 160 Arg Ser Ile Ser Leu Arg Tyr Glu Gly Arg Val Tyr His TyrArg Ile 165 170 175 Asn Thr Ala Ser Asp Gly Lys Leu Tyr Val Ser Ser GluSer Arg Phe 180 185 190 Asn Thr Leu Ala Glu Leu Val His His His Ser ThrVal Ala Asp Gly 195 200 205 Leu Ile Thr Thr Leu His Tyr Pro Ala Pro LysArg Asn Lys Pro Thr 210 215 220 Val Tyr Gly Val Ser Pro Asn Tyr Asp LysTrp Glu Met Glu Arg Thr 225 230 235 240 Asp Ile Thr Met Lys His Lys LeuGly Gly Gly Gln Tyr Gly Glu Val 245 250 255 Tyr Glu Gly Val Trp Lys LysTyr Ser Leu Thr Val Ala Val Lys Thr 260 265 270 Leu Lys Glu Asp Thr MetGlu Val Glu Glu Phe Leu Lys Glu Ala Ala 275 280 285 Val Met Lys Glu IleLys His Pro Asn Leu Val Gln Leu Leu Gly Val 290 295 300 Cys Thr Arg GluPro Pro Phe Tyr Ile Ile Thr Glu Phe Met Thr Tyr 305 310 315 320 Gly AsnLeu Leu Asp Tyr Leu Arg Glu Cys Asn Arg Gln Glu Val Asn 325 330 335 AlaVal Val Leu Leu Tyr Met Ala Thr Gln Ile Ser Ser Ala Met Glu 340 345 350Tyr Leu Glu Lys Lys Asn Phe Ile His Arg Asp Leu Ala Ala Arg Asn 355 360365 Cys Leu Val Gly Glu Asn His Leu Val Lys Val Ala Asp Phe Gly Leu 370375 380 Ser Arg Leu Met Thr Gly Asp Thr Tyr Thr Ala His Ala Gly Ala Lys385 390 395 400 Phe Pro Ile Lys Trp Thr Ala Pro Glu Ser Leu Ala Tyr AsnLys Phe 405 410 415 Ser Ile Lys Ser Asp Val Trp Ala Phe Gly Val Leu LeuTrp Glu Ile 420 425 430 Ala Thr Tyr Gly Met Ser Pro Tyr Pro Gly Ile AspLeu Ser Gln Val 435 440 445 Tyr Glu Leu Leu Glu Lys Asp Tyr Arg Met GluArg Pro Glu Gly Cys 450 455 460 Pro Glu Lys Val Tyr Glu Leu Met Arg AlaCys Trp Gln Trp Asn Pro 465 470 475 480 Ser Asp Arg Pro Ser Phe Ala GluIle His Gln Ala Phe Glu Thr Met 485 490 495 Phe Gln Glu Ser Ser Ile SerAsp Glu Val Glu Lys Glu Leu Gly Lys 500 505 510 Gln Gly Val Arg Gly AlaVal Ser Thr Leu Leu Gln Ala Pro Glu Leu 515 520 525 Pro Thr Lys Thr ArgThr Ser Arg Arg Ala Ala Glu His Arg Asp Thr 530 535 540 Thr Asp Val ProGlu Met Pro His Ser Lys Gly Gln Gly Glu Ser Asp 545 550 555 560 Pro LeuAsp His Glu Pro Ala Val Ser Pro Leu Leu Pro Arg Lys Glu 565 570 575 ArgGly Pro Pro Glu Gly Gly Leu Asn Glu Asp Glu Arg Leu Leu Pro 580 585 590Lys Asp Lys Lys Thr Asn Leu Phe Ser Ala Leu Ile Lys Lys Lys Lys 595 600605 Lys Thr Ala Pro Thr Pro Pro Lys Arg Ser Ser Ser Phe Arg Glu Met 610615 620 Asp Gly Gln Pro Glu Arg Arg Gly Ala Gly Glu Glu Glu Gly Arg Asp625 630 635 640 Ile Ser Asn Gly Ala Leu Ala Phe Thr Pro Leu Asp Thr AlaAsp Pro 645 650 655 Ala Lys Ser Pro Lys Pro Ser Asn Gly Ala Gly Val ProAsn Gly Ala 660 665 670 Leu Arg Glu Ser Gly Gly Ser Gly Phe Arg Ser ProHis Leu Trp Lys 675 680 685 Lys Ser Ser Thr Leu Thr Ser Ser Arg Leu AlaThr Gly Glu Glu Glu 690 695 700 Gly Gly Gly Ser Ser Ser Lys Arg Phe LeuArg Ser Cys Ser Ala Ser 705 710 715 720 Cys Val Pro His Gly Ala Lys AspThr Glu Trp Arg Ser Val Thr Leu 725 730 735 Pro Arg Asp Leu Gln Ser ThrGly Arg Gln Phe Asp Ser Ser Thr Phe 740 745 750 Gly Gly His Lys Ser GluLys Pro Ala Leu Pro Arg Lys Arg Ala Gly 755 760 765 Glu Asn Arg Ser AspGln Val Thr Arg Gly Thr Val Thr Pro Pro Pro 770 775 780 Arg Leu Val LysLys Asn Glu Glu Ala Ala Asp Glu Val Phe Lys Asp 785 790 795 800 Ile MetGlu Ser Ser Pro Gly Ser Ser Pro Pro Asn Leu Thr Pro Lys 805 810 815 ProLeu Arg Arg Gln Val Thr Val Ala Pro Ala Ser Gly Leu Pro His 820 825 830Lys Glu Glu Ala Glu Lys Gly Ser Ala Leu Gly Thr Pro Ala Ala Ala 835 840845 Glu Pro Val Thr Pro Thr Ser Lys Ala Gly Ser Gly Ala Pro Gly Gly 850855 860 Thr Ser Lys Gly Pro Ala Glu Glu Ser Arg Val Arg Arg His Lys His865 870 875 880 Ser Ser Glu Ser Pro Gly Arg Asp Lys Gly Lys Leu Ser ArgLeu Lys 885 890 895 Pro Ala Pro Pro Pro Pro Pro Ala Ala Ser Ala Gly LysAla Gly Gly 900 905 910 Lys Pro Ser Gln Ser Pro Ser Gln Glu Ala Ala GlyGlu Ala Val Leu 915 920 925 Gly Ala Lys Thr Lys Ala Thr Ser Leu Val AspAla Val Asn Ser Asp 930 935 940 Ala Ala Lys Pro Ser Gln Pro Gly Glu GlyLeu Lys Lys Pro Val Leu 945 950 955 960 Pro Ala Thr Pro Lys Pro Gln SerAla Lys Pro Ser Gly Thr Pro Ile 965 970 975 Ser Pro Ala Pro Val Pro SerThr Leu Pro Ser Ala Ser Ser Ala Leu 980 985 990 Ala Gly Asp Gln Pro SerSer Thr Ala Phe Ile Pro Leu Ile Ser Thr 995 1000 1005 Arg Val Ser LeuArg Lys Thr Arg Gln Pro Pro Glu Arg Ile Ala 1010 1015 1020 Ser Gly AlaIle Thr Lys Gly Val Val Leu Asp Ser Thr Glu Ala 1025 1030 1035 Leu CysLeu Ala Ile Ser Arg Asn Ser Glu Gln Met Ala Ser His 1040 1045 1050 SerAla Val Leu Glu Ala Gly Lys Asn Leu Tyr Thr Phe Cys Val 1055 1060 1065Ser Tyr Val Asp Ser Ile Gln Gln Met Arg Asn Lys Phe Ala Phe 1070 10751080 Arg Glu Ala Ile Asn Lys Leu Glu Asn Asn Leu Arg Glu Leu Gln 10851090 1095 Ile Cys Pro Ala Thr Ala Gly Ser Gly Pro Ala Ala Thr Gln Asp1100 1105 1110 Phe Ser Lys Leu Leu Ser Ser Val Lys Glu Ile Ser Asp IleVal 1115 1120 1125 Gln Arg 1130

What is claimed is:
 1. An isolated polypeptide which comprises a mutatedfunctional Abl kinase domain that is resistant to inhibition of itstyrosine kinase activity byN-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-piperazin-1-ylmethyl)-benzamideor a salt thereof.
 2. An isolated polypeptide according to claim 1,wherein the mutated functional Abl kinase domain comprises the aminoacid sequence of the native human Abl kinase domain or an essentiallysimilar sequence thereof in which at least one amino acid is replaced byanother amino acid.
 3. An isolated polypeptide according to claim 2,wherein in the amino acid sequence of the native human Abl kinase domainor an essentially similar sequence thereof at least one amino acidselected from Leu248, Glu255, Lys271, Glu286, Met290, Thr315, Tyr320,Asn322, Glu373, His375 and Ala380 is replaced by another amino acid. 4.An isolated polypeptide according to claim 3, wherein in the amino acidsequence of the native human Abl kinase domain or an essentially similarsequence thereof at least one amino acid selected from Leu248, Glu255,Lys271, Glu286, Met290, Tyr320, Asn322, Glu373, His375 and Ala380 isreplaced by another amino acid.
 5. An isolated polypeptide according toclaim 4, wherein in the amino acid sequence of the native human Ablkinase domain or an essentially similar sequence thereof at least oneamino acid selected from Leu248, Lys271, Glu286, Met290, Tyr320, Asn322,Glu373, His375 and Ala380 is replaced by another amino acid.
 6. Anisolated polypeptide according to claim 3, wherein in the amino acidsequence of the native human Abl kinase domain or an essentially similarsequence thereof at least one amino acid selected from Glu255, Thr315and Ala380 is replaced by another amino acid.
 7. An isolated polypeptideaccording to claim 6, wherein in the amino acid sequence of the nativehuman Abl kinase domain or an essentially similar sequence thereof atleast one amino acid selected from Glu255 and Ala380 is replaced byanother amino acid.
 8. An isolated polypeptide according to any one ofclaims 2 to 7, wherein in the amino acid sequence of the native humanAbl kinase domain or an essentially similar sequence thereof a singleamino acid is replaced by another amino acid.
 9. An isolated polypeptideaccording to claim 7, wherein in the amino acid sequence of the nativehuman Abl kinase domain or an essentially similar sequence thereofGlu255 is replaced by another amino acid.
 10. An isolated polypeptideaccording to claim 3, wherein the amino acid sequence of the nativehuman Abl kinase domain or an essentially similar sequence thereofcontains at least one amino acid mutation selected from Glu255Val,Glu255Lys, Thr315Val, Thr315Leu, Thr315Met, Thr315Gln, Thr315Phe andAla380Thr.
 11. An isolated polypeptide according to claim 10, whereinthe amino acid sequence of the native human Abl kinase domain or anessentially similar sequence thereof contains at least one amino acidmutation selected from Glu255Val, Thr315Val, Thr315Leu, Thr315Met,Thr315Gln, Thr315Phe and Ala380Thr.
 12. An isolated polypeptideaccording to claim 11, wherein the amino acid sequence of the nativehuman Abl kinase domain or an essentially similar sequence thereofcontains at least one amino acid mutation selected from Thr315Leu,Thr315Met, Thr315Gln and Thr315Phe.
 13. An isolated polypeptideaccording to any one of claims 10 to 12, wherein the amino acid sequenceof the native human Abl kinase domain or an essentially similar sequencethereof contains a single amino acid mutation.
 14. An isolatedpolypeptide according to claim 11, wherein the amino acid sequence ofthe native human Abl kinase domain or an essentially similar sequencethereof contains the amino acid mutation Glu255Val.
 15. An isolatedpolypeptide according to any one of claims 2 to 14, wherein the aminoacid sequence of the native human Abl kinase domain consists of aminoacids 229-500 of SEQ ID NO:
 2. 16. An isolated polypeptide according toany one of claims 2 to 15, which is a Bcr-Abl tyrosine kinase.
 17. Useof a polypeptide according to any one of claims 2 to 16 to screen forcompounds which inhibit the tyrosine kinase activity of saidpolypeptide.
 18. An isolated nucleic acid molecule comprising anucleotide sequence that encodes a polypeptide according to any one ofclaims 2 to
 16. 19. Use of a nucleic acid molecule according to claim 18in the production of a polypeptide according to any one of claims 2 to16 for use in screening for compounds which inhibit the tyrosine kinaseactivity of said polypeptide.
 20. A recombinant vector comprising anucleic acid molecule according to claim
 18. 21. A recombinant vectoraccording to claim 20, which is a recombinant expression vector.
 22. Ahost cell comprising a recombinant vector according to claim 20 or 21.